PROJECT SUMMARY The lymphatic system is critical for tissue fluid homeostasis, lipid absorption, and immune surveillance and is no longer considered a ?passive bystander? in pathological processes. The digestive lymphatic system, includ- ing intestinal and mesenteric lymphatics, plays a critical role in several pathological processes including obe- sity, metabolic disease, and dyslipidemia in addition to lymphedema and inflammation.3,4 While prenatal lym- phatic development has been an area of intensive investigation, insights into factors involved in post-natal reg- ulation of lymphatic maturation, remodeling, and maintenance of lymphatic structure and function is limited. Our laboratory has recently shown that the receptor tyrosine kinase TIE1 is required for lymphatic remodeling and normal lymphatic function in utero, and that deletion of Tie1 specifically from the developing lymphatic vas- culature prior to birth results in abnormal LV formation. We present new preliminary data that deletion of Tie1 in the early post-natal period results in malnutrition with poor weight gain, abnormal LV maturation, and asci- tes. In contrast, we show that Tie1 deletion in the adult animal results in severe LV dysfunction and profound diet-induced obesity (DIO) when challenged with a high fat diet (HFD). We hypothesize that TIE1 cell auton- omous and non-cell autonomous signaling plays a unique role in orchestrating early post-natal LV and lacteal maturation and function, and that TIE1 signaling is essential for maintenance of LV and lacteal structure and function in the mature animal. Therefore, we propose to: 1) Delineate the specific role of TIE1 signaling in early post-natal lacteal and LV maturation, and nutrient absorption, in vivo. Tie1fl/fl mice and an inducible LEC Cre line, Prox1CreERT, will be used to produce LEC-specific, temporal deletion of Tie1 (Tie1lecKO) in post-natal lymphatics. Quantitative and functional analyses will be used to characterize the unique phenotypes that result from TIE1 attenuation in intestinal lacteals and mesenteric LVs. 2) Determine the role of TIE1 in maintenance of lymphatic function in the adult and characterize its role in mediating DIO. Tie1lecKO and control (Tie1fl/fl) adult mice will be place on a 60% HFD or normal chow for 6 weeks following exposure to Tamoxifen. LV structure and function will be measured as well as alterations in body weight and composition, levels of circulating hormones insulin, leptin and adiponectin, sensitivity to glucose and insulin, and adipocyte morphology and inflammatory status. 3) Define the mechanisms of TIE1-mediated signaling required for LV and lacteal remodeling and maintenance, in vivo. Utilizing CRISPR/Cas9, we have devel- oped mice with missense mutations in critical intracellular domains of Tie1. Unique domain-specific effects on LV and lacteal structure and function will be characterized in the early post-natal and adult animals during the dynamic reorganization and maintenance processes, respectively. scRNA-seq and sc-UniFrac analysis will be employed to define TIE1-dependent signaling required for cell autonomous and non-autonomous LEC subtype transcriptional signatures regulating maturation and maintenance of the GI lymphatic system after birth.